Substituted 2-(2,6 dioxo-3-fluoropiperidin-3-yl)-isoindolines and method of reducing TNFα levels

ABSTRACT

1-Oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindolines and 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindolines reduce the levels of TNFα in a mammal. A typical embodiment is 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline.

The present invention relates to2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindolines, the method ofreducing levels of tumor necrosis factor α in a mammal through theadministration thereof, and pharmaceutical compositions of suchderivatives.

BACKGROUND OF THE INVENTION

Tumor necrosis factor α, or TNFα, is a cytokine which is releasedprimarily by mononuclear phagocytes in response to a numberimmunostimulators. When administered to animals or humans, it causesinflammation, fever, cardiovascular effects, hemorrhage, coagulation,and acute phase responses similar to those seen during acute infectionsand shock states. Excessive or unregulated TNFα production thus has beenimplicated in a number of disease conditions. These include endotoxemiaand/or toxic shock syndrome {Tracey et al., Nature 330, 662-664 (1987)and Hinshaw et al., Circ. Shock 30, 279-292 (1990)}; cachexia {Dezube etal., Lancet, 335 (8690), 662 (1990)} and Adult Respiratory DistressSyndrome where TNFα concentration in excess of 12,000 pg/mL have beendetected in pulmonary aspirates from ARDS patients {Millar et al.,Lancet 2(8665), 712-714 (1989)}. Systemic infusion of recombinant TNFαalso resulted in changes typically seen in ARDS {Ferrai-Baliviera etal., Arch. Surg. 124(12), 1400-1405 (1989)}.

TNFα appears to be involved in bone resorption diseases, includingarthritis. When activated, leukocytes will produce bone-resorption, anactivity to which the data suggest TNFα contributes. {Bertolini et al.,Nature 319, 516-518 (1986) and Johnson et al., Endocrinology 124(3),1424-1427 (1989).} TNFα also has been shown to stimulate bone resorptionand inhibit bone formation in vitro and in vivo through stimulation ofosteoclast formation and activation combined with inhibition ofosteoblast function. Although TNFα may be involved in many boneresorption diseases, including arthritis, the most compelling link withdisease is the association between production of TNFα by tumor or hosttissues and malignancy associated hypercalcemia {Calci. Tissue Int. (US)46(Suppl.), S3-10 (1990)}. In Graft versus Host Reaction, increasedserum TNFα levels have been associated with major complication followingacute allogenic bone marrow transplants {Holler et al., Blood, 75(4),1011-1016 (1990)}.

Cerebral malaria is a lethal hyperacute neurological syndrome associatedwith high blood levels of TNFα and the most severe complicationoccurring in malaria patients. Levels of serum TNFα correlated directlywith the severity of disease and the prognosis in patients with acutemalaria attacks {Grau et al., N. Engl. J. Med. 320(24), 1586-1591(1989)}.

Macrophage-induced angiogenesis TNFα is known to be mediated by TNFα.Leibovich et al. {Nature, 329, 630-632 (1987)} showed TNFα induces invivo capillary blood vessel formation in the rat cornea and thedeveloping chick chorioallantoic membranes at very low doses and suggestTNFα is a candidate for inducing angiogenesis in inflammation, woundrepair, and tumor growth. TNFα production also has been associated withcancerous conditions, particularly induced tumors {Ching et al., Brit.J. Cancer, (1955) 72, 339-343, and Koch, Progress in MedicinalChemistry, 22, 166-242 (1985)).

TNFα also plays a role in the area of chronic pulmonary inflammatorydiseases. The deposition of silica particles leads to silicosis, adisease of progressive respiratory failure caused by a fibroticreaction. Antibody to TNFα completely blocked the silica-induced lungfibrosis in mice {Pignet et al., Nature, 344, 245-247 (1990)}. Highlevels of TNFα production (in the serum and in isolated macrophages)have been demonstrated in animal models of silica and asbestos inducedfibrosis {Bissonnette et al., Inflammation 13(3), 329-339 (1989)}.Alveolar macrophages from pulmonary sarcoidosis patients have also beenfound to spontaneously release massive quantities of TNFα as comparedwith macrophages from normal donors {Baughinan et al., J. Lab. Clin.Med. 115(1), 36-42 (1990)}.

TNFα is also implicated in the inflammatory response which followsreperfusion, called reperfusion injury, and is a major cause of tissuedamage after loss of blood flow {Vedder et al., PNAS, 87, 2643-2646(1990)}. TNFα also alters the properties of endothelial cells and hasvarious pro-coagulant activities, such as producing an increase intissue factor pro-coagulant activity and suppression of theanticoagulant protein C pathway as well as down-regulating theexpression of thrombomodulin (Sherry et al., J. Cell Biol 107, 1269-1277(1988)}. TNFα has pro-inflammatory activities which together with itsearly production (during the initial stage of an inflammatory event)make it a likely mediator of tissue injury in several importantdisorders including but not limited to, myocardial infarction, strokeand circulatory shock. Of specific importance may be TNFα-inducedexpression of adhesion molecules, such as intercellular adhesionmolecule (ICAM) or endothelial leukocyte adhesion molecule (ELAM) onendothelial cells {Munro et al., Am. J. Path. 135(1), 121-132 (1989)}.

TNFα blockage with monoclonal anti-TNFα antibodies has been shown to bebeneficial in rheumatoid arthritis {Elliot et al., Int. J. Pharmac. 199517(2), 141-145). High levels of TNFα are associated with Crohn's disease{von Dullemen et al., Gastroenterology, 1995 109(1), 129-135} andclinical benefit has been acheived with TNFα antibody treatment.

Moreover, it now is known that TNFα is a potent activator of retrovirusreplication including activation of HIV-1. (Duh et al., Proc. Nat. Acad.Sci. 86, 5974-5978 (1989); Poll et al., Proc. Nat. Acad. Sci. 87,782-785 (1990); Monto et al., Blood 79, 2670 (1990); Clouse et al., J.Immunol. 142, 431-438 (1989); Poll et al., AIDS Res. Hum. Retrovirus,191-197 (1992)}. AIDS results from the infection of T lymphocytes withHuman Immunodeficiency Virus (HIV). At least three types or strains ofHIV have been identified, i.e., HIV-1, HIV-2 and HIV-3. As a consequenceof HIV infection, T-cell mediated immunity is impaired and infectedindividuals manifest severe opportunistic infections and/or unusualneoplasms. HIV entry into the T lymphocyte requires T lymphocyteactivation. Other viruses, such as HIV-1, HIV-2 infect T lymphocytesafter T cell activation and such virus protein expression and/orreplication is mediated or maintained by such T cell activation. Once anactivated T lymphocyte is infected with HIV, the T lymphocyte mustcontinue to be maintained in an activated state to permit HIV geneexpression and/or HIV replication. Cytokines, specifically TNFα, areimplicated in activated T-cell mediated HIV protein expression and/orvirus replication by playing a role in maintaining T lymphocyteactivation. Therefore, interference with cytokine activity such as byprevention or inhibition of cytokine production, notably TNFα, in anHIV-infected individual assists in limiting the maintenance of Tlymphocyte caused by HIV infection.

Monocytes, macrophages, and related cells, such as kupffer and glialcells, also have been implicated in maintenance of the HIV infection.These cells, like T cells, are targets for viral replication and thelevel of viral replication is dependent upon the activation state of thecells. {Rosenberg et al., The Immunopathogenesis of HIV Infection,Advances in Immunology, 57 (1989)}. Cytokines, such as TNFα, have beenshown to activate HIV replication in monocytes and/or macrophages {Poliet al., Proc. Natl. Acad. Sci., 87, 782-784 (1990)}, therefore,prevention or inhibition of cytokine production or activity aids inlimiting HIV progression for T cells. Additional studies have identifiedTNFα as a common factor in the activation of HIV in vitro and hasprovided a clear mechanism of action via a nuclear regulatory proteinfound in the cytoplasm of cells (Osbom, et al., PNAS 86 2336-2340). Thisevidence suggests that a reduction of TNFα synthesis may have anantiviral effect in HIV infections, by reducing the transcription andthus virus production.

AIDS viral replication of latent HIV in T cell and macrophage lines canbe induced by TNFα {Folks et al., PNAS 86, 2365-2368 (1989)}. Amolecular mechanism for the virus inducing activity is suggested byTNFα's ability to activate a gene regulatory protein (NFκB) found in thecytoplasm of cells, which promotes HIV replication through binding to aviral regulatory gene sequence (LTR) {Osborn et al., PNAS 86, 2336-2340(1989)}. TNFα in AIDS associated cachexia is suggested by elevated serumTNFα and high levels of spontaneous TNFα production in peripheral bloodmonocytes from patients {Wright et al., J. Immunol. 141(1), 99-104(1988)}. TNFα has been implicated in various roles with other viralinfections, such as the cytomegalia virus (CMV), influenza virus,adenovirus, and the herpes family of viruses for similar reasons asthose noted.

The nuclear factor κB (NFκB) is a pleiotropic transcriptional activator(Lenardo, et al., Cell 1989, 58, 227-29). NFκB has been implicated as atranscriptional activator in a variety of disease and inflammatorystates and is thought to regulate cytokine levels including but notlimited to TNFα and also to be an activator of HIV transcription(Dbaibo, et al., J. Biol. Chem. 1993, 17762-66; Duh et al., Proc. Natl.Acad. Sci. 1989, 86, 5974-78; Bachelerie et al., Nature 1991, 350,709-12; Boswas et al., J. Acquired Immune Deficiency Syndrome 1993, 6,778-786; Suzuki et al., Biochem. And Biophys. Res. Comm. 1993, 193,277-83; Suzuki et al., Biochem. And Biophys. Res Comm. 1992, 189,1709-15; Suzuki et al., Biochem. Mol. Bio. Int. 1993, 31(4); 693-700;Shakhov et al., Proc. Natl. Acad. Sci. USA 1990, 171, 35-47; and Staalet al., Proc. Natl. Acad. Sci. USA 1990, 87, 9943-47). Thus, inhibitionof NFκB binding can regulate transcription of cytokine gene(s) andthrough this modulation and other mechanisms be useful in the inhibitionof a multitude of disease states. The compounds described herein caninhibit the action of NFκB in the nucleus and thus are useful in thetreatment of a variety of diseases including but not limited torheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, otherarthritic conditions, septic shock, septis, endotoxic shock, graftversus host disease, wasting, Crohn's disease, ulcerative colitis,multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, HIV,AIDS, and opportunistic infections in AIDS. TNFα and NFκB levels areinfluenced by a reciprocal feedback loop. As noted above, the compoundsof the present invention affect the levels of both TNFα and NFκB.

Many cellular functions are mediated by levels of adenosine 3',5'-cyclic monophosphate (cAMP). Such cellular functions can contributeto inflammatory conditions and diseases including asthma, inflammation,and other conditions (Lowe and Cheng, Drugs of the Future, 17(9),799-807, 1992). It has been shown that the elevation of cAMP ininflammatory leukocytes inhibits their activation and the subsequentrelease of inflammatory mediators, including TNFα and NFκB. Increasedlevels of cAMP also leads to the relaxation of airway smooth muscle.Phosphodiesterases control the level of cAMP through hydrolysis andinhibitors of phosphodiesterases have been shown to increase cAMPlevels.

Decreasing TNFα levels and/or increasing cAMP levels thus constitutes avaluable therapeutic strategy for the treatment of many inflammatory,infectious, immunological or malignant diseases. These include but arenot restricted to septic shock, sepsis, endotoxic shock, hemodynamicshock and sepsis syndrome, post ischemic reperfusion injury, malaria,mycobacterial infection, meningitis, psoriasis, congestive heartfailure, fibrotic disease, cachexia, graft rejection, cancer, autoimmunedisease, opportunistic infections in AIDS, rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, other arthritic conditions,Crohn's disease, ulcerative colitis, multiple sclerosis, systemic lupuserythrematosis, ENL in leprosy, radiation damage, and hyperoxic alveolarinjury. Prior efforts directed to the suppression of the effects of TNFαhave ranged from the utilization of steroids such as dexamethasone andprednisolone to the use of both polyclonal and monoclonal antibodies{Beutler et al., Science 234, 470-474 (1985); WO 92/11383 }.

DETAILED DESCRIPTION

The present invention is based on the discovery that certain classes ofnon-polypeptide compounds more fully described herein decrease thelevels of TNFα , increase cAMP levels, and inhibit phosphodiesterase.

In particular, the invention pertains to

(a) a 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of the formula:##STR1## in which Y is oxygen or H₂ and

each of R¹, R², R³, and R⁴, independently of the others, is hydrogen,halo, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, oramino, and

(b) the acid addition salts of said2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindolines which contain anitrogen atom capable of being protonated.

Unless otherwise defined, the term alkyl denotes a univalent saturatedbranched or straight hydrocarbon chain containing from 1 to 8 carbonatoms. Representative of such alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, and tert-butyl. Alkoxy refers toan alkyl group bound to the remainder of the molecule through anethereal oxygen atom. Representative of such alkoxy groups are methoxy,ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, andtert-butoxy.

The compounds of Formula I are used, under the supervision of qualifiedprofessionals, to inhibit the undesirable effects of TNFα and inhibitphosphodiesterase. The compounds can be administered orally, rectally,or parenterally, alone or in combination with other therapeutic agentsincluding antibiotics, steroids, etc., to a mammal in need of treatment.

The compounds of the present invention also can be used topically in thetreatment or prophylaxis of topical disease states mediated orexacerbated by excessive TNFα production, respectively, such as viralinfections, such as those caused by the herpes viruses, or viralconjunctivitis, psoriasis, atopic dermatitis, etc.

The compounds also can be used in the veterinary treatment of mammalsother than humans in need of prevention or inhibition of TNFαproduction. TNFα mediated diseases for treatment, therapeutically orprophylactically, in animals include disease states such as those notedabove, but in particular viral infections. Examples include felineimmunodeficiency virus, equine infectious anaemia virus, caprinearthritis virus, visna virus, and maedi virus, as well as otherlentiviruses.

The compounds of Formula I are readily prepared through a number ofroutes. In a first embodiment, the ring nitrogen of a2-(2,6-dioxopiperidin-3-yl)-isoindoline of Formula II is protected witha conventional amino protecting group to yield a protected2-(2,6-dioxopiperidin-3-yl)-isoindoline of Formula III. This is thenfluorinated to yield a protected2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of Formula IV,following which removal of the protecting group yields the compounds ofFormula V: ##STR2##

In the foregoin reactions, each of R¹, R², R³, R⁴, and Y are as definedabove and X is amino protecting group. When any of R¹, R², R³, and R⁴ isamino, it too should be protected prior to the fluorination step.

Some of the compounds of Formula II which are here utilized asintermediates are known, as for example1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline and1,3-dioxo-2-(2, 6-dioxopiperidin-3 -yl)-5-aminoisoindoline. See, e.g.,Jonsson, Acta Pharma. Succica, 9, 521-542 (1972). Others are describedin copending application Ser. No. 08/701,494, U.S. Pat. No. 5,798,368the disclosure of which is incorporated herein by reference, or can beprepared by methods analogous thereto.

The fluorination can be effected with a variety of reagents, as forexample, N-fluorobenzenesulfonimide, perchloryl fluoride,N-fluorobenzenedisulfonimide, and the like, in the presence of a strongbase such as n-butyl lithium, sodium hydride, lithium diisopropylamide,and the like.

In a second method, an appropriately substituted glutamic acid diesterof Formula VI is fluorinated to yield the correstponding fluoroglutamicacid diester of Formula VI. This is then converted to the fluorinatedglutamic acid anhydride of Formula VIII which, in turn, is amidated toyield the compounds of Formula V: ##STR3##

In the foregoin reactions, each of R¹, R², R³, R⁴, and Y are as definedabove and Z and Z' are lower alkyl. Again when any of R¹, R², R³, and R⁴is amino, it too should be protected prior to the fluorination step.

Protecting groups utilized herein denote groups which generally are notfound in the final therapeutic compounds but which are intentionallyintroduced at some stage of the synthesis in order to protect groupswhich otherwise might be altered in the course of chemicalmanipulations. Such protecting groups are removed at a later stage ofthe synthesis and compounds bearing such protecting groups thus are ofimportance primarily as chemical intermediates (although somederivatives also exhibit biological activity). Accordingly the precisestructure of the protecting group is not critical. Numerous reactionsfor the formation and removal of such protecting groups are described ina number of standard works including, for example, "Protective Groups inOrganic Chemistry", Plenum Press, London and N.Y., 1973; Greene, Th. W."Protective Groups in Organic Synthesis", Wiley, N.Y. 1981; "ThePeptides", Vol. I, Schroder and Lubke, Academic Press, London and N.Y.,1965; "Methoden der organischen Chemie", Houben-Weyl, 4th Edition,Vol.15/I, Georg Thieme Verlag, Stuttgart 1974, the disclosures of whichare incorporated herein by reference.

In any of the foregoing reactions, a nitro compound can be employed withthe nitro group being converted to an amino group by catalytichydrogenation. Alternatively, a protected amino group can be cleaved toyield the corresponding amino compound. An amino group can be protectedas an amide utilizing an acyl group which is selectively removable undermild conditions, especially benzyloxycarbonyl, formyl, or a loweralkanoyl group which is branched in 1- or α position to the carbonylgroup, particularly tertiary alkanoyl such as pivaloyl, a lower alkanoylgroup which is substituted in the position α to the carbonyl group, asfor example trifluoroacetyl.

The carbon atom to which the depicted fluorine atom is bound in thecompounds of Formula I constitutes a center of chirality, thereby givingrise to optical isomers: ##STR4##

Both the racemates of these isomers and the individual isomersthemselves, as well as diastereomers when a second chiral center ispresent, are within the scope of the present invention. The racematescan be used as such or can be separated into their individual isomersmechanically as by chromatography using a chiral absorbant.Alternatively, the individual isomers can be prepared in chiral form orseparated chemically from a mixture by forming salts with a chiral acidor base, such as the individual enantiomers of 10-camphorsulfonic acid,camphoric acid, α-bromocamphoric acid, methoxyacetic acid, tartaricacid, diacetyltartaric acid, malic acid, pyrrolidone-5-carboxylic acid,and the like, and then freeing one or both of the resolved bases,optionally repeating the process, so as obtain either or bothsubstantially free of the other; i.e., in a form having an opticalpurity of >95%.

The present invention also pertains to the physiologically acceptablenon-toxic acid addition salts of the compound of Formula I which containa group capable of being protonated; e.g., amino. Such salts includethose derived from organic and inorganic acids such as, withoutlimitation, hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid, methanesulphonic acid, acetic acid, tartaric acid, lacticacid, succinic acid, citric acid, malic acid, maleic acid, sorbic acid,aconitic acid, salicylic acid, phthalic acid, embonic acid, enanthicacid, and the like.

Particularly preferred compounds include1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-aminoisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-methylisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-methylisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-methylisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-methylisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethylisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-aminoisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethylisoindoline,and1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline.Of these, 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline and1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline are particularlypreferred.

Oral dosage forms include tablets, capsules, dragees, and similarshaped, compressed pharmaceutical forms containing from 1 to 100 mg ofdrug per unit dosage. Isotonic saline solutions containing from 20 to100 mg/mL can be used for parenteral administration which includesintramuscular, intrathecal, intravenous and intra-arterial routes ofadministration. Rectal administration can be effected through the use ofsuppositories formulated from conventional carriers such as cocoabutter.

Pharmaceutical compositions thus comprise one or more compounds ofFormulas I associated with at least one pharmaceutically acceptablecarrier, diluent or excipient. In preparing such compositions, theactive ingredients are usually mixed with or diluted by an excipient orenclosed within such a carrier which can be in the form of a capsule orsachet. When the excipient serves as a diluent, it may be a solid,semi-solid, or liquid material which acts as a vehicle, carrier, ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, elixirs, suspensions, emulsions,solutions, syrups, soft and hard gelatin capsules, suppositories,sterile injectable solutions and sterile packaged powders. Examples ofsuitable excipients include lactose, dextrose, sucrose, sorbitol,mannitol, starch, gum acacia, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidinone, cellulose, water, syrup, and methylcellulose, the formulations can additionally include lubricating agentssuch as talc, magnesium stearate and mineral oil, wetting agents,emulsifying and suspending agents, preserving agents such as methyl- andpropylhydroxybenzoates, sweetening agents or flavoring agents.

The compositions preferably are formulated in unit dosage form, meaningphysically discrete units suitable as a unitary dosage, or apredetermined fraction of a unitary dose to be administered in a singleor multiple dosage regimen to human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with a suitablepharmaceutical excipient. The compositions can be formulated so as toprovide an immediate, sustained or delayed release of active ingredientafter administration to the patient by employing procedures well knownin the art.

Enzyme-linked immunosorbent assays for TNFα can be performed in aconventional manner. PBMC is isolated from normal donors byFicoll-Hypaque density centrifugation. Cells are cultured in RPMIsupplemented with 10% AB+ serum, 2 mM L-glutamine, 100 U/mL penicillin,and 100 mg/mL streptomycin. Drugs are dissolved in dimethylsulfoxide(Sigma Chemical) and further dilutions are done in supplemented RPML.The final dimethylsulfoxide concentration in the presence or absence ofdrug in the PBMC suspensions is 0.25 wt %. Drugs are assayed at half-logdilutions starting at 50 mg/mL. Drugs are added to PBMC (10⁶ cells/mL)in 96 wells plates one hour before the addition of LPS. PBMC (10⁶cells/mL) in the presence or absence of drug are stimulated by treatmentwith 1 mg/mL of LPS from Salmonella minnesota R595 (List BiologicalLabs, Campbell, Calif.). Cells are then incubated at 37° C. for 18-20hours. Supernatants are harvested and assayed immediately for TNFαlevels or kept frozen at -70° C. (for not more than 4 days) untilassayed. The concentration of TNFα in the supernatant is determined byhuman TNFα ELISA kits (ENDOGEN, Boston, Mass.) according to themanufacturer's directions.

Phosphodiesterase can be determined in conventional models. For example,using the method of Hill and Mitchell, U937 cells of the humanpromonocytic cell line are grown to 1×10⁶ cells /mL and collected bycentrifugation. A cell pellet of 1×10⁹ cells is washed in phosphatebuffered saline and then frozen at -70° C. for later purification orimmediately lysed in cold homogenization buffer (20 mM Tris-HCl, pH 7.1,3 mM 2-mercaptoethanol, 1 mM magnesium chloride, 0.1 mM ethyleneglycol-bis-(β3-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 μMphenylmethylsulfonyl fluoride (PMSF), and 1 μg/mL leupeptin). Cells arehomogenized with 20 strokes in a Dounce homogenizer and supernatantcontaining the cytosolic fraction are obtained by centrifugation. Thesupernatant then is loaded onto a Sephacryl S-200 column equilibrated inhomogenization buffer. Phosphodiesterase is eluted in homogenizationbuffer at a rate of approximately 0.5 mL/min and fractions are assayedfor phosphodiesterase activity -/+ rolipram. Fractions containingphosphodiesterase activity (rolipram sensitive) are pooled and aliquotedfor later use.

The phosphodiesterase assay is carried out based on procedure describedby Hill and Mitchell. The assay is carried out in a total volume of 100μl containing various concentration of the test compounds, 50 mMTris-HCl, pH 7.5, 5 mM magnesium chloride and 1 μM cAMP of which 1% was³ H cAMP. Reactions are incubated at 30° C. for 30 minutes andterminated by boiling for 2 minutes. The amount of phosphodiesterase IVcontaining extract used for these experiments is predetermined such thatreactions are within the linear range and consumed less than 15% of thetotal substrate. Following termination of reaction, samples are chilledat 4° C. and then treated with 10 μl 10 mg/mL snake venom for 15 min at30° C. Unused substrate then is removed by adding 200 μl of a quaternaryammonium ion exchange resin (AG1-X8, BioRad) for 15 minutes. Samplesthen are spun at 3000 rpm, 5 min and 50 μl of the aqueous phase aretaken for counting. Each data point is carried out in duplicate andactivity is expressed as percentage of control. The IC₅₀ of the compoundthen is determined from dose response curves of a minimum of threeindependent experiments.

The following examples will serve to further typify the nature of thisinvention but should not be construed as a limitation in the scopethereof, which scope is defined solely by the appended claims.

EXAMPLE 1

To a stirred suspension of1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindoline (2.0 g, 7.75 mmol) anddi-tert.-butyl dicarbonate (1.86 g, 8.52 mmol) in 1,4-dioxane (3.0 mL)is added dimethylaminopyridine (1.00 mg) at room temperature. Thesolution is stirred at room temperature for 18 hours and the solventthen removed in vacuo. The residue is stirred with ether (30 mL) for 30minutes, filtered, and washed with ether to give1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)isoindoline.

EXAMPLE 2

Similarly to the procedure of Example 1, there are respectively obtainedfrom1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)- 4,5,6,7-tetrachloroisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethylisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-isoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)- 4,5,6,7-tetrachloroisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)- 4,5,6,7-tetramethylisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-methylisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-5-methylisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-methylisoindoline, and1-oxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline, thecompounds1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline,1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline,1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethylisoindoline,1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline,1-oxo-2-(1-tert. -butoxycarbonyl-2,6-dioxopiperidin-3-yl)-isoindoline, 1-oxo-2-(1 -tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline,1 -oxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline,1 -oxo-2-(1 -tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethylisoindoline,1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4-methylisoindoline,1,3 -dioxo-2-(1 -tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-5-methylisoindoline, 1-oxo-2-(1 -tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-5-methylisoindoline,1-oxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4-methylisoindoline,and1-oxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline

Similarly utilizing equivalent amounts of1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline,1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline,1-oxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline, and1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline, but utilizing 3.72g of di-tert.-butyl dicarbonate in the procedure of Example 1, there arerespectively obtained1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4-(1-tert.-butoxycarbonylamino)-isoindoline,1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-5-(1-tert.-butoxycarbonylamino)-isoindoline,1-oxo-2-(1 -tert. -butoxycarbonyl-2,6-dioxopiperidin-3-yl)-5-(1-tert.-butoxycarbonylamino)-isoindoline, and1-oxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4-(1-tert.-butoxycarbonylamino)-isoindoline.

EXAMPLE 3

To a stirred solution of1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)isoindoline(1.0 g, 2.8 mmol) in tetrahydrofuran (10 mL) is added n-butyl lithium(1.2 mL, 3.0 mmol, 2.5M) at -78° C. After 20 minutes,N-fluorobenzenesulfonimide (0-8 g, 3.2 mmol) is added to the mixture.The mixture is allowed to reach room temperature and the solvent thenremoved in vacuo. The residue is stirred with ethyl acetate (10 mL) andIN hydrochloric acid (10 mL) for one hour. The organic layer isseparated and the solvent removed in vacuo to yield1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline which can befurther purified through chromatography.

EXAMPLE 4

The procedure of Example 3 is followed, substituting, however, 0.76 g ofN-fluorobenzenedisulfonimide for the 0.8 g ofN-fluorobenzenesulfonimide. There is thereby obtained1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline which can befurther purified through column chromatography.

EXAMPLE 5

To a stirred solution of1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)isoindoline(1.0 g. 2.8 mmol) in tetrahydrofuran (10 mL) is added lithiumdiisopropylamide (1.5 mL, 3.0 mmol, 2M) at . After 30 minutes,perchloryl fluoride (5 mmol) is bubbled into the mixture. The mixture isallowed to reach room temperature and the solvent then removed in vacuo.The residue is stirred with ethyl acetate (10 mL) and IN hydrochloricacid (10 mL) for one hour. The organic layer is separated and thesolvent removed in vacuo to yield1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline which can befurther purified through chromatography.

EXAMPLE 6

To a stirred solution of1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-isoindoline(1.0 g. 2.8 mmol) in dimethylformamide (10 mL) is added sodium hydride(112 mg, 2.8 mmol, 60%) at room temperature. After about 30 minutes,perchloryl fluoride (5 mmol) is bubbled into the mixture . The mixtureis stirred with methylene chloride (10 mL) and IN hydrochloric acid (10mL) for one hour. The organic layer is separated 30 and the solventremoved in vacuo to yield1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline which can befurther purified through chromatography.

EXAMPLE 7

To a stirred solution of1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)isoindoline(1.0 g, 2.8 mmol) and tetramethylethylene diamine (0.5 g, 4.3 mmol) intetrahydrofuran (10 mL) is added n-butyl lithium (1.2 mL, 3.0 mmol, 2.5M) at -78° C. After 30 minutes, N-fluorobenzenesulfonimide (0-8 g, 3.2mmol) is added to the mixture. The mixture is allowed to reach roomtemperature and the solvent then removed in vacuo. The residue isstirred with ethyl acetate (10 mL) and IN hydrochloric acid (10 mL) forone hour. The organic layer is separated and the solvent removed invacuo to yield 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindolinewhich can be further purified through column chromatography.

EXAMPLE 8

Starting with each of1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4-(1-tert.-butoxycarbonylamino)-isoindoline,1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-5-(1-tert.-butoxycarbonylamino)-isoindoline,1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline,1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline,1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethylisoindoline,1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline,1-oxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-5-(1-tert.-butoxycarbonylamino)-isoindoline,1-oxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-isoindoline,1-oxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4-(1-tert.-butoxycarbonylamino)-isoindoline,1-oxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline,1-oxo-2-(l-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline,1-oxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethylisoindoline, 1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3 -yl)-4-methylisoindoline,1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-5-methylisoindoline,1-oxo-2-(1-tert.- butoxycarbonyl-2,6-dioxopiperidin- 3-yl)-5-methylisoindoline,1-oxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4-methylisoindoline, and1-oxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline,there are respectively obtained by following the procedures of Examples3, 4, 5, 6, or 7,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-aminoisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethylisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-aminoisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethylisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-methylisoindoline,1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-methylisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-methylisoindoline,1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-methylisoindoline, and1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline.

EXAMPLE 9

Part A.

A solution of L-glutamic acid dimethyl ester (2.6 g, 14.8 mmol), isoamylnitrite (2.13 mL, 15.9 mmol) and acetic acid (0.22 mL) in benzene (150mL) is heated to reflux for one hour. The solution is washed with INaqueous sulfuric acid, water, saturated sodium hydrogen carbonatesolution, water and brine (50 mL each). The solvent is removed in vacuoto yield dimethyl 2-diazopentane-1,5-dioate which can be furtherpurified by column chromatography.

Part B.

To a cold solution of 5 mL of 70% hydrogen fluoride in pyridine and 1.2g (6.7 mmol) N-bromosuccinimide in 10 mL of ether is added a solution ofdimethyl 2-diazopentane-1,5-dioate (1.1 g, 5.9 mmol) in ether (10 mL) at0C. The mixture is stirred at 0° C. for 30 minutes The solution iswashed with water (20 mL), brine (20 mL) and dried over sodium sulfate.The solvent is removed in vacuo to yield dimethyl2-bromo-2-fluoropentane-1,5-dioate which can be further purified bycolumn chromatography.

Part C.

A mixture of dimethyl 2-bromo-2-fluoropentane-1,5-dioate (1.0 g, 3.8mmol) and potassium phthalimide (0.79 g, 4.3 mmol) in dimethylformamide(10 mL) is heated at 80° C. for 3 hours. The solvent is removed in vacuoand the residue is stirred with ethyl acetate (50 mL) for 10 minutes.The organic layer is washed with water and brine (20 mL each), and driedover sodium sulfate. The solvent is removed in vacuo to yield dimethyl2-(1,3-dioxoisoindolin-2-yl)-2-fluoropenta-1,5-dioate which can befurther purified by column chromatography.

Part D.

A mixture of dimethyl2-(1,3-dioxoisoindolin-2-yl)-2-fluoropenta-1,5-dioate (1.3 g, 4.0 mmol),methanol (10 mL) and 4N hydrochloric acid (10 mL) is heated at 80° C. orone hour. The solvent is removed in vacuo to yield2-fluoro-2-(1,3-dioxoisoindolin-2-yl)-propane-1,3-dicarboxylic acid.This is dissolved in acetic anhydride (20 mL) and the solution heated atreflux for 30 minutes. The solvent is remove in vacuo to yield2-fluoro-2-(1,3-dioxoisoindolin-2-yl)-propane-1,3-dicarboxylic acidanhydride which is mixed with ammonia in methanol (35 mL, 2M) andstirred at room temperature for 18 hours. The solvent is then removed invacuo and the residue is stirred with methylene chloride (50 mL) for 10minutes. The organic layer is washed with water and brine (40 mL each),and dried over sodium sulfate. The solvent is removed in vacuo and theresidue is heated at reflux with carbonyl diimidazole (0.65 g. 4 mmol)and dimethylaminopyridine (50 mg) in tetrahydrofuran (30 mL) for 18hours. 1,3-Dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline isisolated extraction with ethyl acetate and further purified bychromatography.

EXAMPLE 10

A stirred mixture of L-glutamic acid dimethyl ester (2.0 g, 11.4 mmol)and phthalic anhydride (1.7 g, 11.4 mmol) in acetic acid (30 mL) isheated to refluxed for one hour. The solvent is removed in vacuo toyield dimethyl 2-(1,3-dioxoisoindolin-2-yl)-pentane-1,5-dioate which isfurther purified through chromatography.

To a stirred solution of dimethyl2-(1,3-dioxoisoindolin-2-yl)-pentane-1,5-dioate (1.0 g, 3.3 mmol) andtetramethylethylene diamine (0.5 g, 4.3 mmol) in tetrahydrofuran (10 mL)is added 2.5M n-butyl lithium (1.6 mL, 4 mmol,) at -79° C. After 30minutes, N-fluorobenzenesulfonimide (1 g, 3.2 mmol) is added to themixture which then is allowed to reach room temperature. The solvent isremoved in vacuo and the residue is stirred with methylene chloride (100mL) for 10 minutes. The organic layer is washed with water and brine (30mL each), and dried over sodium sulfate. The solvent is removed in vacuoto yield dimethyl2-(1,3-dioxoisoindolin-2-yl)-2-fluoropentane-1,5-dioate which is furtherpurified by chromatography and converted to1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline as describedabove in Part D of Example 9.

EXAMPLE 11

A stirred mixture of ethyl bromofluoroacetate (1.0 g, 5.4 mmol) andpotassium phthalide (1.0 g, 5.4 mmol) in dimethylformamide (10 mL) isheated at 80° C. for 3 hours. The mixture is stirred with ether (50 mL)and water (50 mL) and the organic layer then washed with water and brine(30 mL each), and dried over sodium sulfate. The solvent is removed invacuo to give ethyl 2-(1,3-dioxoisoindolin-2-yl)-2-fluoroacetate whichis further purified through chromatography.

To a stirred solution of ethyl2-(1,3-dioxoisoindolin-2-yl)-2-fluoroacetate (0.80 g, 3.2 mmol) intetrahydrofuran (30 mL) is added lithium diisopropylamide (1.7 mL, 3.4mmol, 2M) at -78° C. After 30 minutes, t-butyl acrylate (0.42 g, 3.2mmol) is added to the mixture which is allowed to reach roomtemperature. The solvent is removed in vacuo and the residue stirredwith methylene chloride (50 mL) and water (30 mL) for 10 min. Theorganic layer is washed with brine (30 mL), and dried over sodiumsulfate. The solvent is removed in vacuo to give tert-butyl4-(1,3-dioxoisoindolin-2-yl)-4-fluoro-4-ethoxycarbonylbutanoate which isfurther purified by column chromatography.

A solution of tert-butyl4-(1,3-dioxoisoindolin-2-yl)-4-fluoro-4-ethoxycarbonylbutanoate (1.1 g,3 mmol) and trifluoroacetic acid (5 mL) in methylene chloride (5 mL) isstirred for 18 hours and then with methylene chloride (50 mL) for 10min. The organic layer is washed with water and brine (30 mL each), anddried over sodium sulfate. The solvent is removed in vacuo to yield4-(1,3-dioxoisoindolin-2-yl)-4-(ethoxycarbonyl)-4-fluorobutanoic acidwhich can be purified by chromatography or used in the next step withoutfurther purification.

A mixture of4-(1,3-dioxoisoindolin-2-yl)-4-(ethoxycarbonyl)-4-fluorobutanoic acid(0.9 g, 2.8 mmol), carbonyl diimidazole (0.46 g, 2.8 mmol) anddimethylaminopyrimidine (0.68 g, 5.6 mmol) in tetrahydrofuran (30 mL) isheated at reflux for 18 hours. The solvent is removed in vacuo and theresidue is stirred with methylene chloride (50 mL) for 10 minutes. Theorganic layer is washed with water and brine (40 mL each) and dried oversodium sulfate. The solvent is removed in vacuo to give1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline which isfurther purified by column chromatography.

EXAMPLE 12

Tablets, each containing 50 mg of1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline, can beprepared in the following manner:

    ______________________________________    Constituents (for 1000 tablets)    ______________________________________    1,3-dioxo-2-(2,6-dioxo-                           50.0 g    3-fluoropiperidin-3-yl)-    isoindoline    lactose                50.7 g    wheat starch            7.5 g    polyethylene glycol 6000                            5.0 g    talc                    5.0 g    magnesium stearate      1.8 g    demineralized water    q.s.    ______________________________________

The solid ingredients are first forced through a sieve of 0.6 mm meshwidth. The active ingredient, lactose, talc, magnesium stearate and halfof the starch then are mixed. The other half of the starch is suspendedin 40 mL of water and this suspension is added to a boiling solution ofthe polyethylene glycol in 100 mL of water. The resulting paste is addedto the pulverulent substances and the mixture is granulated, ifnecessary with the addition of water. The granulate is dried overnightat 35° C., forced through a sieve of 1.2 mm mesh width and compressed toform tablets of approximately 6 mm diameter which are concave on bothsides.

EXAMPLE 13

Tablets, each containing 100 mg of1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline, can be preparedin the following manner:

    ______________________________________    Constituents (for 1000 tablets)    ______________________________________    1-oxo-2-(2,6-dioxo-    100.0 g    3-fluoropiperidin-3-yl)-    isoindoline    lactose                100.0 g    wheat starch            47.0 g    magnesium stearate      3.0 g    ______________________________________

All the solid ingredients are first forced through a sieve of 0.6 mmmesh width. The active ingredient, lactose, magnesium stearate and halfof the starch then are mixed. The other half of the starch is suspendedin 40 mL of water and this suspension is added to 100 mL of boilingwater. The resulting paste is added to the pulverulent substances andthe mixture is granulated, if necessary with the addition of water. Thegranulate is dried overnight at 35° C., forced through a sieve of 1.2 mmmesh width and compressed to form tablets of approximately 6 mm diameterwhich are concave on both sides.

EXAMPLE 14

Tablets for chewing, each containing 75 mg of1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline, can be preparedin the following manner:

    ______________________________________    Composition (for 1000 tablets)    ______________________________________    1-oxo-2-(2,6-dioxo-     75.0 g    3-fluoropiperidin-3-yl)-    isoindoline    mannitol               230.0 g    lactose                150.0 g    talc                    21.0 g    glycine                 12.5g    stearic acid            10.0g    saccharin               1.5 g    5% gelatin solution    q.s.    ______________________________________

All the solid ingredients are first forced through a sieve of 0.25 mmmesh width. The mannitol and the lactose are mixed, granulated with theaddition of gelatin solution, forced through a sieve of 2 mm mesh width,dried at 50° C. and again forced through a sieve of 1.7 mm mesh width.1-Oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline, the glycine andthe saccharin are carefully mixed, the mannitol, the lactose granulate,the stearic acid and the talc are added and the whole is mixedthoroughly and compressed to form tablets of approximately 10 mmdiameter which are concave on both sides and have a breaking groove onthe upper side.

EXAMPLE 15

Tablets, each containing 10 mg1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline,can be prepared in the following manner:

    ______________________________________    Composition (for 1000 tablets)    ______________________________________    1,3-dioxo-2-(2,6-dioxo-  10.0 g    3-fluoropiperidin-3-yl)-    4,5,6,7-tetrafluoroisoindoline    lactose                 328.5 g    corn starch              17.5 g    polyethylene glycol 6000                             5.0 g    talc                     25.0 g    magnesium stearate       4.0 g    demineralized water     q.s.    ______________________________________

The solid ingredients are first forced through a sieve of 0.6 mm meshwidth. Then the active imide ingredient, lactose, talc, magnesiumstearate and half of the starch are intimately mixed. The other half ofthe starch is suspended in 65 mL of water and this suspension is addedto a boiling solution of the polyethylene glycol in 260 mL of water. Theresulting paste is added to the pulverulent substances, and the whole ismixed and granulated, if necessary with the addition of water. Thegranulate is dried overnight at 35° C., forced through a sieve of 1.2 mmmesh width and compressed to form tablets of approximately 10 mmdiameter which are concave on both sides and have a breaking notch onthe upper side.

EXAMPLE 16

Gelatin dry-filled capsules, each containing 100 mg of1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline, can beprepared in the following manner:

    ______________________________________    Composition (for 1000 capsules)    ______________________________________    1-oxo-2-(2,6-dioxo-    100.0 g    3-fluoropiperidin-3-yl)-    5-aminoisoindoline    microcrystalline cellulose                            30.0 g    sodium lauryl sulfate   2.0 g    magnesium stearate      8.0 g    ______________________________________

The sodium lauryl sulfate is sieved into the1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline through asieve of 0.2 mm mesh width and the two components are intimately mixedfor 10 minutes. The microcrystalline cellulose is then added through asieve of 0.9 mm mesh width and the whole is again intimately mixed for10 minutes. Finally, the magnesium stearate is added through a sieve of0.8 mm width and, after mixing for a further 3 minutes, the mixture isintroduced in portions of 140 mg each into size 0 (elongated) gelatindry-fill capsules.

EXAMPLE 17

A 0.2% injection or infusion solution can be prepared, for example, inthe following manner:

    ______________________________________    1,3-dioxo-2-(2,6-dioxo-                            5.0 g    3-fluoropiperidin-3-yl)-    5-aminoisoindoline hydrochloride    sodium chloride         22.5 g    phosphate buffer pH 7.4                           300.0 g    demineralized water    to 2500.0 mL    ______________________________________

1,3-Dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindolinehydrochloride is dissolved in 1000 mL of water and filtered through amicrofilter. The buffer solution is added and the whole is made up to2500 mL with water. To prepare dosage unit forms, portions of 1.0 or 2.5mL each are introduced into glass ampoules (each containing respectively2.0 or 5.0 mg of imide).

What is claimed is:
 1. A compound selected from the group consistingof(a) a 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of the formula:##STR5## in which Y is oxygen or H₂ andeach of R¹, R², R³, and R⁴,independently of the others, is hydrogen, halo, alkyl of 1 to 4 carbonatoms, alkoxy of 1 to 4 carbon atoms, or amino, and (b) the acidaddition salts of said 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindolineswhich contain a nitrogen atom capable of being protonated.
 2. Thecompound according to claim 1 in which R¹, R², R³, and R⁴ are hydrogen.3. A compound according to claim 1 in which R¹, R², R³, and R⁴ are thesame and each is chloro, fluoro, methyl, or methoxy.
 4. A compoundaccording to claim 1 in which R³ is amino, and R', R², and R⁴ arehydrogen.
 5. A compound according to claim 1 in which R⁴ is amino, andR¹, R², and R³ are hydrogen.
 6. A compound according to claim 1 in whichR³ is methyl, and R¹, R², and R⁴ are hydrogen.
 7. A compound accordingto claim 1 in which R⁴ is methyl, and R¹, R², and R³ are hydrogen. 8.The compound according to claim 1 which is1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline.
 9. Thecompound according to claim 1 which is1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-aminoisoindoline. 10.The compound according to claim 1 which is1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline. 11.The compound according to claim 1 which is1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline.12. The compound according to claim 1 which is1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline.
 13. The compound according to claim 1which is 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethylisoindoline.
 14. The compound according to claim 1which is1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline.15. The compound according to claim 1 which is1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline.
 16. Thecompound according to claim 1 which is1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline.
 17. The compoundaccording to claim 1 which is1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-aminoisoindoline.
 18. Thecompound according to claim 1 which isl-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline.19. The compound according to claim 1 which is1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline.20. The compound according to claim 1 which is1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethylisoindoline.21. The compound according to claim 1 which is1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline22. The method of reducing undesirable levels of TNFα in a mammal whichcomprises administering thereto an effective amount of a compoundaccording to claim
 1. 23. A pharmaceutical composition comprising aquantity of a compound according to claim 1 sufficient uponadministration in a single or multiple dose regimen to reduce levels ofTNFα in a mammal in combination with a carrier.